Bottom Line:
In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before.Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin.Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III.

ABSTRACTVenoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.

toxins-07-04468-f001: Electrophoretic separation of 2 CPLL-treated honeybee venom samples. On the left, venom from queen honeybees was collected; on the right, venom from winter worker honeybees was collected. CPLL flow-through (=FT) and elution (=EL) samples are separated on a 10% Tris-glycine-SDS-PAGE gel (A) and a 16.5% Tris-tricine-SDS-PAGE gel (B). Molecular weight regions that are known to contain high amounts of PLA2 (*) and melittin (►) are indicated. Molecular weights (in kDa) of the markers are indicated in the figure: (A) PageRuler Prestained Protein Ladder; (B) Spectra Multicolor Low Range Protein Ladder.

Mentions:
The in-depth proteomic analysis of the venom from winter workers of A. mellifera revealed 656 unique tryptic peptides (see Supplementary Tables S1 and S2), providing biological evidence for 88 venom proteins and peptides. Queen venom, on the other hand, revealed 521 unique tryptic peptides (see Supplementary Tables S1 and S2), providing biological evidence for 76 venom proteins and peptides. In a preceding study, honeybee venom compounds were categorized into the groups of putative toxins and venom trace molecules by prediction of their biological function and subcellular location [16]. Since less abundant compounds were enriched by the CPLL pretreatment (Figure 1), a subgroup of identified proteins was found that probably only have a local function in the venom duct or reservoir or are released by leakage of the gland tissue. This group of venom trace molecules is fully listed in Supplementary Table S3. At present, no function could be attributed to 16 compounds. They were categorized separately (Supplementary Table S4) because they lacked functional domains and/or similar annotated sequences. In contrast to venom trace molecules, venom toxins are typically highly abundant and are actively secreted by the venom glands to contribute to the venom defense or social immunity function. In total, 34 toxins are listed in Table 1, complemented by the compounds that are also secreted but show no clear toxic venom function and therefore are categorized under venom trace molecules. Of these 34 putative venom toxins, two had not been discovered in honeybee venom before (serine proteinase stubble and antithrombin-III). Despite the identification of new venom toxins, some of the previously reported honeybee venom compounds were missing from our list. The antigen 5-like wasp venom paralog was previously proven to show seasonal variation since it could be expressed by venom gland tissue of winter bees but not of summer bees [16]. However, this antigen 5-like venom protein could not be detected by our FT-ICR MS studies in any of the three (summer worker, winter worker, queen) venom samples, which could be due to venom sample variation or technological variation (liquid versus gel-based proteomics). Other honeybee venom compounds that have been described long ago (like cardiopep and minimine) together with some small peptides (like tertiapin and apidaecin) were lacking in the list of toxins found by FTMS in summer worker venom [18]. Yet, the present study also failed to identify these compounds using the same techniques on venom from honeybee queen and winter workers. However, vitellogenin, one of the venom compounds that was lacking in the MS study on summer worker venom, could now be detected in venom from A. mellifera queens.

toxins-07-04468-f001: Electrophoretic separation of 2 CPLL-treated honeybee venom samples. On the left, venom from queen honeybees was collected; on the right, venom from winter worker honeybees was collected. CPLL flow-through (=FT) and elution (=EL) samples are separated on a 10% Tris-glycine-SDS-PAGE gel (A) and a 16.5% Tris-tricine-SDS-PAGE gel (B). Molecular weight regions that are known to contain high amounts of PLA2 (*) and melittin (►) are indicated. Molecular weights (in kDa) of the markers are indicated in the figure: (A) PageRuler Prestained Protein Ladder; (B) Spectra Multicolor Low Range Protein Ladder.

Mentions:
The in-depth proteomic analysis of the venom from winter workers of A. mellifera revealed 656 unique tryptic peptides (see Supplementary Tables S1 and S2), providing biological evidence for 88 venom proteins and peptides. Queen venom, on the other hand, revealed 521 unique tryptic peptides (see Supplementary Tables S1 and S2), providing biological evidence for 76 venom proteins and peptides. In a preceding study, honeybee venom compounds were categorized into the groups of putative toxins and venom trace molecules by prediction of their biological function and subcellular location [16]. Since less abundant compounds were enriched by the CPLL pretreatment (Figure 1), a subgroup of identified proteins was found that probably only have a local function in the venom duct or reservoir or are released by leakage of the gland tissue. This group of venom trace molecules is fully listed in Supplementary Table S3. At present, no function could be attributed to 16 compounds. They were categorized separately (Supplementary Table S4) because they lacked functional domains and/or similar annotated sequences. In contrast to venom trace molecules, venom toxins are typically highly abundant and are actively secreted by the venom glands to contribute to the venom defense or social immunity function. In total, 34 toxins are listed in Table 1, complemented by the compounds that are also secreted but show no clear toxic venom function and therefore are categorized under venom trace molecules. Of these 34 putative venom toxins, two had not been discovered in honeybee venom before (serine proteinase stubble and antithrombin-III). Despite the identification of new venom toxins, some of the previously reported honeybee venom compounds were missing from our list. The antigen 5-like wasp venom paralog was previously proven to show seasonal variation since it could be expressed by venom gland tissue of winter bees but not of summer bees [16]. However, this antigen 5-like venom protein could not be detected by our FT-ICR MS studies in any of the three (summer worker, winter worker, queen) venom samples, which could be due to venom sample variation or technological variation (liquid versus gel-based proteomics). Other honeybee venom compounds that have been described long ago (like cardiopep and minimine) together with some small peptides (like tertiapin and apidaecin) were lacking in the list of toxins found by FTMS in summer worker venom [18]. Yet, the present study also failed to identify these compounds using the same techniques on venom from honeybee queen and winter workers. However, vitellogenin, one of the venom compounds that was lacking in the MS study on summer worker venom, could now be detected in venom from A. mellifera queens.

Bottom Line:
In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before.Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin.Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III.

ABSTRACTVenoms of invertebrates contain an enormous diversity of proteins, peptides, and other classes of substances. Insect venoms are characterized by a large interspecific variation resulting in extended lists of venom compounds. The venom composition of several hymenopterans also shows different intraspecific variation. For instance, venom from different honeybee castes, more specifically queens and workers, shows quantitative and qualitative variation, while the environment, like seasonal changes, also proves to be an important factor. The present study aimed at an in-depth analysis of the intraspecific variation in the honeybee venom proteome. In summer workers, the recent list of venom proteins resulted from merging combinatorial peptide ligand library sample pretreatment and targeted tandem mass spectrometry realized with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS/MS). Now, the same technique was used to determine the venom proteome of queens and winter bees, enabling us to compare it with that of summer bees. In total, 34 putative venom toxins were found, of which two were never described in honeybee venoms before. Venom from winter workers did not contain toxins that were not present in queens or summer workers, while winter worker venom lacked the allergen Api m 12, also known as vitellogenin. Venom from queen bees, on the other hand, was lacking six of the 34 venom toxins compared to worker bees, while it contained two new venom toxins, in particularly serine proteinase stubble and antithrombin-III. Although people are hardly stung by honeybees during winter or by queen bees, these newly identified toxins should be taken into account in the characterization of a putative allergic response against Apis mellifera stings.